We have developed an immunohistochemical technique, based on the reduction of EF5-(2-(2-nitro-1H-imidazol-1-yI)-N(2,2,3,3,3-pentafluoropropyl) acetamide), to evaluate the presence and distribution of hy poxia in vivo. The results of the first 5 human patients treated with EF5 are presented. Patients with head and neck squamous cell carcinoma (SCC; n=2), cervical SCC (n=2) and soft tissue sarcoma (n=1) were treated with 9 mg/kg EF5 2 days preceding tumor surgery. EF5 related toxicities were not seen over 28 days of follow-up. EF5 half life was 13 hrs and 50% of the parent drug was recovered from urine over 48 hrs. In vitro incubation of cell lines from all 5 tumors with EF5 demonstrated consistency of binding between cell types and a sigmoid relationship between binding and po2 over 0.1% - 10% oxygen. Incubation of freshly obtained tissue cubes in 0.5% oxygen and 600 uM hours EF3 showed a factor of 2 between the maximal binding of 4 different SCC tumors. Despite the consistency of binding rate of the tissues and cells, the in situ binding (in vivo binding during the 48 hrs preceding surgery) varied by a factor of >12 between the highest and lowest binding SCC. In vitro binding studies using tumor cubes and cells show minimal variation in the cells/tissues' ability to reduce and bind EF5. Substantial inter-tumoral in situ heterogeneity of hypoxia, as documented by EF5 binding with immunohistochemistry is seen between human tumor types with similar histologies. Dose escalation and studies in other tumor sites are proceeding.